In vivo iron labeling of stem cells and tracking these labeled stem cells after their transplantation

ABSTRACT

Intravenous ferumoxytol is used to effectively label mesenchymal stem cells (MSCs) in vivo and is used for in vivo tracking of stem cell transplants with magnetic resonance (MR) imaging. The method eliminates risk of contamination and biologic alteration of MSCs associated with ex-vivo-labeling procedures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication 61/755,283 filed Jan. 22, 2013, which is incorporated hereinby reference.

STATEMENT OF GOVERNMENT SPONSORED SUPPORT

This invention was made with Government support under contract no.2R01AR054458-05 awarded by the National Institutes of Health (NIH). TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to in vivo iron labeling of stem cells andsubsequent tracking of these stem cells with magnetic resonance imagingafter their transplantation.

BACKGROUND OF THE INVENTION

Each year, arthritis results in 44 million outpatient visits, 992100hospitalizations, and 700000 knee replacement procedures(www.cdc.gov/arthritis). The need for knee replacement is rapidlyincreasing, with 3.48 million expected procedures by 2030. However,artificial implants are associated with potential complications, such asperiprosthetic fractures, loosening, and metal sensitivity. Even in theabsence of complications, the lifetime of an artificial prosthesis islimited to approximately 10 years as the implant wears out.

Cell transplants, particularly stem cell-scaffold nanocomposites, couldovercome these problems by providing long-term biologic restoration ofjoint defects. Bone marrow-derived mesenchymal stem cells (MSCs) havebeen established as a promising source for stem cell-mediated jointrepair in a clinical setting. MSCs can be obtained with a bone marrowaspirate, are expanded in vitro, and can differentiate into all jointcomponents. However, interactions between transplanted MSCs and thepatient's host environment are still poorly understood.

To monitor successful engraftment and recognize complications such asgraft failure or tumor formation, MSC therapies require tracking of thetransplanted stem cells. In the past, stem cell tracking has beenachieved on the basis of the concept of ex vivo contrast agent labeling.This approach requires multiple ex vivo manipulations of stem cellsbetween their harvest and transplantation.

Clinical translation of ex vivo-labeling procedures is complicated froma regulatory point of view as these manipulations greatly enhance therisk of cell sample contamination, alterations in stem cell biology, orin vivo side effects from added transfection agents. Most transfectionagents (LIPOFECTAMINE 2000 [Invitrogen, Carlsbad, Calif.] orpoly-L-lysine [Sigma-P4707; Sigma-Aldrich, St Louis, Mo.]) are not U.S.Food and Drug Administration (FDA) approved. In addition, someultra-small super-paramagnetic iron oxide-transfection agentcombinations have induced cytotoxic effects or altered the stem cellbiology.

Accordingly, the art is in need of more immediately clinicallyapplicable methods for stem cell labeling, which would not require exvivo manipulations of harvested cells and which would eliminate the needfor transfection agents, that then could be used to track transplantedMSCs. The present invention addresses this need.

SUMMARY OF THE INVENTION

Instead of conventional labeling ex vivo in cell culture, the method ofthis invention is an in vivo labeling method of mesenchymal stem cells(MSCs) with intravenous injection of ferumoxytol (Feraheme; AMAGPharmaceuticals, Lexington, Mass.), a Food and Drug Administration(FDA)-approved intravenous iron supplement. The in vivo iron labeling oruptake by the stem cells is a result of phagocytosis or endocytosisfollowing the intravenous injection. With in vivo labeling, iron uptakewas found to be superior to comparative ex vivo labeling, and labeled,collected MSCs that were subsequently implanted into the knees of ratswith an induced osteochondral defect could be readily detected onT2-weighted MR images for at least 4 weeks after transplantation.

Specifically, a clinical stem cell therapy method is provided for invivo and noninvasively monitoring of stem cell implants. Iron oxidenanoparticles are intravenously injected into a subject to achieve invivo phagocytotic (or endocytotic) iron labeling of stem cells (e.g.mesenchymal stem cells). In one example, the subject is a human and theiron oxide nanoparticles are dosed at 28 mg Fe/kg. In another example,the subject is an animal (e.g. a rodent) and the iron oxidenanoparticles are dosed at 5-10 mg/kg for the subject. It is noted thatthis labeling or uptake of iron by the stem cells occurs in vivo withoutany addition of a transfection agent. It is also noted that the in vivolabeling does not use any ex vivo labeling or ex vivo manipulations tothe stem cells.

After one to three days upon the intravenous injection, iron-labeledstem cells are harvested from the bone marrow of the subject. In oneexample, directly after the harvesting the harvested iron-labeled stemcells are transplanted into the same subject or a different subject. Inanother example, the harvested iron-labeled stem cells can be expandedex vivo. The transplantation in that case will then take place three tofour weeks after the ex vivo expansion. Transplantation could take placein an organ, or specifically in a joint, a brain, a heart, a liver, or apancreas.

In vivo and noninvasively monitoring of the transplanted stem cellsusing magnetic resonance imaging can now take place to determine theaccuracy of stem cell transplants, the immediate engraftment pattern,and the long-term retention at the target allows us to optimize stemcell treatment protocols. The in vivo labeling would eliminate safetyconcerns associated with ex vivo stem cell manipulations and enable invivo detection of lost or rejected stem cell transplants early enoughfor corrective actions.

While the specific embodiment pertains to a mechanical osteochondraldefect as the therapeutic target, it is important to realize that thisis just an example application. Being able to MR track iron-labeled MSCssafely and effectively will have myriad clinical applications to avariety of other stem cell transplants in other target organs, includingbut not limited to stroke, myocardial infarct, and a range of autoimmunediseases including multiple sclerosis and type I diabetes mellitus.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent of application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a flow diagram of the clinical stem cell therapy methodaccording to an exemplary embodiment of the invention.

FIG. 2 shows according to an exemplary embodiment of the invention invivo ferumoxytol labeling of bone marrow mesenchymal stem cells (MSCs)and subsequent in vivo tracking of transplanted MSCs with MR imaging.The method relies on intravenous injection of ferumoxytol (red arrow210) and its phagocytosis by cells in the reticuloendothelial system(liver, spleen, and bone marrow). Ferumoxytol-labeled bone marrow cellsare harvested by using bone marrow aspiration (red square insets 220)and in this example are expanded in vitro for 7 days. Inset 230: Cellsseeded on a scaffold. The labeled cells are then seeded in an agarosescaffold and implanted in an osteochondral defect of the distal femur.Sagittal T2-weighted MR image 240 (repetition time msec/echo time msec,4000/15, 30, 45, 60) shows monitoring of transplant engraftment (arrow242).

FIG. 3 shows according to an exemplary embodiment of the invention, thatthe majority of in vivo labeled cells, that had been harvested from bonemarrow and expanded in MSC-selective culture for 7 days, are MSC. CD105(one of several MSC markers) and CD68 (macrophage marker) stains of bonemarrow-derived cells after 7 days of MSC-selective culture. Evaluationof 3000 4′,6-diamidine-2-phenylindole-positive cells in 12 high-powerfields (magnification, 320) revealed a mean of 70.2%+/−1.9 MSCs(CD105-positive cells=green) and of 13.5%+/−2.3 macrophages(CD68-positive cells=red) per high-power field.4′,6-Diamidine-2-phenylindole (blue) counterstain visualized cell nucleiof all cells in these samples, including CD105- and CD68-positive cellsand other cells. hpf=High-power field.

FIG. 4 shows according to an exemplary embodiment of the inventionferumoxytol uptake by MSCs, labeled in vivo via ferumoxytol injection orlabeled ex vivo via protamine transfection. 3,3′Diaminobenzidine(DAB)-Prussian blue staining, fluorescence microscopy, confocalmicroscopy, and corresponding 3D fluorescent plots show intracellulariron uptake for in vivo- and ex vivo-labeled cells, with a highereffectiveness of the in vivo-labeling technique compared with ex vivolabeling. Transmission electron microscopy images showcompartmentalization of iron oxide nanoparticles (arrows) in secondarylysosomes in in vivo- and ex vivo-labeled cells, with relatively higherintralysosomal nanoparticle quantities in the in vivo-labeled cells.Nonlabeled control cells are shown for comparison.4′,6-Diamidine-2-phenylindole (blue), rhodamine (red), and FITC (green)signals represent nuclei, cytoskeleton, and iron nanoparticles,respectively. Colocalization of red and green signals indicates thepresence of iron nanoparticles in the cytoplasm. This colocallization ofgreen and red signals results in yellow signal for high concentrationsof iron nanoparticles in in vivo-labeled cells and orange color forsmaller concentrations of iron nanoparticles in ex vivo-labeled cells.The 3D plots reveal the amount of FITC-positive signal (ironnanoparticles) as peaks per pixel in each cell.

FIGS. 5A-C show according to an exemplary embodiment of the inventionlongitudinal in vitro evaluations of FITC-ferumoxytol-labeled MSCs. InFIG. 5A, fluorescence microscopy demonstrates green fluorescence signalof in vivo FITC-ferumoxytol-labeled cells. The fluorescence signalslowly declines over time. Corresponding T2 relaxation time maps of cellpellets in test tubes show shortening of T2 relaxation times of invivo-labeled cell pellets compared with unlabeled control pellets, whichalso decreases slowly over time. Color spectrum=color scale for T2 time(milliseconds) that signifies MR signal intensity of the pellet; themore iron (ferumoxytol) the cells contain, the lower the T2 value.LIV=labeled in vivo, C=control cells. FIG. 5B shows quantitative T2relaxation times of in vivo-labeled cell pellets and unlabeled controlcells at weeks 1, 2, 3, and 4 after extraction from bone marrow showshortening of T2 relaxation times of in vivo-labeled cell pelletscompared with unlabeled control cells. FIG. 5C shows iron content percell of in vivo-labeled cells, ex vivo-labeled cells, and control cells,as measured by inductively coupled plasma optical emission spectrometry.In vivo-labeled cells show significantly higher iron uptake at day 7compared with ex vivo-labeled cells and control cells. Data aredisplayed as means and standard errors (error bars) of triplicatesamples per experimental group, with 4×10⁵ cells per sample.

FIG. 6 shows according to an exemplary embodiment of the invention thatin vivo-labeled MSCs maintain their chondrogenic differentiationpotential in vitro. MSC pellets after differentiation in chondrogenicmedia for 4 weeks maintain a 3D structure, as shown by hematoxylin-eosin(H & E) stains. Alcian blue- and safranin O-positive stains confirmchondrogenic matrix production for both in vivo-labeled cells andcontrol cells.

FIGS. 7A-B show according to an exemplary embodiment of the inventionthat in vivo labeled MSC can be detected on sagittal T2-weighted MRimages (4000/15, 30, 45, 60) after implantation in osteochondral defectsof rat knee joints. In FIG. 7A, representative T2 relaxation time mapsshow shortened T2 relaxation times of in vivo-labeled MSC transplants(arrow) compared with unlabeled control transplants (arrowhead) at weeks0, 2, and 4 after transplantation. The T2 signal effect of labeledtransplants slowly decreases over time. Color spectrum=color scale forT2 time (milliseconds) that signifies MR signal intensity of theimplant; the more iron (ferumoxytol) the implanted cells contain, thelower the T2 value. In FIG. 7B, corresponding T2 relaxation times showsignificantly shorter T2 values of in vivo-labeled transplants comparedwith unlabeled control transplants up to 4 weeks after implantation.Data are displayed as means and standard errors (error bars) of sixanimals in each group.

FIG. 8 shows according to an exemplary embodiment of the inventionhistopathologic correlation of matrix-associated stem cell implants.Representative sections at 2 weeks and 4 weeks after implantation areshown. Hematoxylin-eosin (H & E) stains show engraftment of allimplants, and 3,3′ diaminobenzidine (DAB)-Prussian blue stains showimplanted cells containing iron (brown staining) at 2 and 4 weeks forlabeled in vivo transplants, whereas unlabeled control transplantsremain unstained. Alcian blue stains show cartilage formation in allimplants.

FIG. 9 shows according to an exemplary embodiment of the inventionnanoparticle characterization by energy dispersive x-ray spectroscopy.High magnification transmission electron microscopy images confirmingpresence of iron nanoparticles in in vivo and ex vivo labeled lysosomesand corresponding energy dispersive spectrums showing higher ironcontent in in vivo labeled samples.

FIG. 10 shows according to an exemplary embodiment of the inventionCD105 and CD68 stains of cell implants in osteochondral defects. At week2 after implantation, there are many CD105 positive MSCs noted in theimplant. At week 4, the number of CD105 positive MSC in the implantslightly decreases, consistent with death and initiated differentiationof some of the transplanted cells, while few CD68 macrophages appear inthe implant.

DETAILED DESCRIPTION

The approach of the method of the present invention (FIG. 1) relies onintravenous administration of an FDA-approved iron supplementferumoxytol (Feraheme; Advanced Magnetics, Cambridge, Mass.) to a stemcell donor prior to stem cell harvest from bone marrow. Ferumoxytol iscomposed of iron oxide nanoparticles, which are taken up by thereticuloendothelial system in vivo, and which provide a strong signalintensity effect on magnetic resonance (MR) images. Accordingly,intravenously injected ferumoxytol would be taken up by mesenchymal stemcells (MSCs) in bone marrow, would be retained in the cells throughharvesting (and in one example ex vivo expansion) and allow forsensitive in vivo MSC detection with MR imaging after transplantationinto osteochondral defects. The method provided herein demonstratesintravenous ferumoxytol administration as a clinically applicable ironsupplement to effectively label MSCs in vivo and for tracking of stemcell transplants.

Materials and Methods

In Vivo MSC Labeling

Sixteen 6-8-week-old Sprague-Dawley rats (Charles River, Wilmington,Mass.) served as MSC donors: Seven rats remained untreated, while ninerats were injected intravenously with ferumoxytol (n=7) or fluoresceinisothiocyanate (FITC) (Fisher Scientific, Pittsburgh, Pa.) conjugatedferumoxytol (n=2) (hereafter referred to as FITC-ferumoxytol) at a doseof 28 mg of iron per kilogram. The details for synthesis are includedinfra in section Synthesis of FITC-conjugated ferumoxytol. This dose hadbeen shown to elicit significant MR signal intensity effects of the bonemarrow in rodents in previous studies. Seven athymic Sprague-Dawley ratsserved as MSC recipients and underwent MR imaging up to 4 weeks afterstem cell transplantation.

MSC Extraction and Cultivation

Donor Sprague-Dawley rats were euthanized by means of CO₂ inhalation 2days after intravenous ferumoxytol injection to allow sufficient timefor phagocytosis by reticuloendothelial system cells, considering ablood half-life of 67 minutes in rodents. Both femurs and tibias wereisolated. The epiphyses were removed, and the bone marrow was flushedwith Dulbecco's modified Eagle's medium (Invitrogen), supplemented with10% fetal bovine serum (Invitrogen). The cells were separated on a cellstrainer (BD, Franklin Lakes, N.J.) to prevent adding any coagulatedtissue in the culture flask, centrifuged at 1800 rpm for 10 minutes andresuspended in 1 mL of ammonium-chloride-potassium buffer (ACK LysingBuffer; Invitrogen) for 2 minutes, washed with phosphate-bufferedsaline, and spun again at 1800 rpm for 10 minutes. The cells were platedin a flask with a 75-cm² flask area (culture surface area) in fullmedia, supplemented with 50 pg of fibroblast growth factor (Gibco,Gaithersburg, Md.) and maintained at 37° C. with 5% CO₂ for 7 days FIG.2). The medium was replaced every 72 hours or when cells reachedconfluence. Nonadherent hematopoietic stem cells, red blood cells, andwhite blood cells were eliminated with every change in culture medium,leaving the adherent and expanding MSCs behind. This direct adherencemethod has shown improved efficiencies for MSC selection, compared withdensity gradient centrifugation.

All in vivo experiments were performed with cells at passage 0 (day 7 oflabeling). In vitro studies involved evaluation of ferumoxytol-labeledcells and unlabeled cells until day 28, corresponding to passage 0-6.Viability assays were performed at each passage by using the trypan blueexclusion test with the use of an automatic cell counter (Countess;Invitrogen).

MSC Immunostaining

Day 7 cells were fixed with 10% formalin (BDH, West Chester, Pa.) andplated on chamber slides at a concentration of cells of 60 000/cm².Immunohistochemical stains against CD105 for MSC (Endoglin M-20; SantaCruz Biotechnology, Dallas, Tex.) and CD68 for macrophages (Abcam,Cambridge, Mass.) were performed, and slides were counterstained byusing 49,6-diamidine-2-phenylindole. Two researchers counted the numberof CD105- and CD68-positive cells separately, and data were averagedover 12 high-power fields (magnification, 320) for each stain.

Evaluation of Ferumoxytol Uptake by MSCs

MSCs labeled with FITC-ferumoxytol in vivo or FITC-conjugatedferumoxytol and protamine (hereafter referred to asFITC-ferumoxytol-protamine) ex vivo, as well as untreated control cells,were evaluated for the presence or absence of green FITC fluorescence byusing a fluorescence microscope (Olympus BH-2; Scion, Frederick, Md.)and image processing software (Metamorph; Molecular Devices, Sunnyvale,Calif.). Cell samples were also analyzed by using confocal microscopy(LSM 510; Carl Zeiss, Thornwood, N.Y.). Fluorescence intensities andthree-dimensional 3D) intensity plots were calculated with ImageJsoftware (http://rsbweb.nih.gov/ij/) by using an established protocoland a threshold of 20 fluorescence units.

To evaluate the compartmentalization of iron oxide nanoparticles inMSCs, 400000 cells (in triplicate) labeled in vivo with ferumoxytol,labeled ex vivo with ferumoxytol and protamine, or untreated (controlcells) were processed for electron microscopy. Sections of 100-nmthickness of resin-embedded cell samples were placed on 100-meshFormvar-coated copper grids (FCF2010-Cu; Electron Microscopy Sciences,Hatfield, Pa.) and imaged using a transmission electron microscope(Tecnai F20 X-Twin; FEI, Hillsboro, Oreg.).

In addition, triplicate samples of in vivo-labeled MSCs (at days 7 and14), ex vivo-labeled MSCs, and unlabeled control cells underwentinductively coupled plasma optical emission spectrometry forquantification of intracellular iron content. The iron content persample was divided by cell concentration to provide iron content percell.

Evaluation of in Vitro MR Signal Intensity Effects ofFerumoxytol-Labeled MSCs

Triplicate samples of 400000 labeled and unlabeled control cells at days7, 14, 21, and 28 after extraction were suspended in 10 mL of agarosescaffold (Sigma-Aldrich) and underwent MR imaging with a 7-T animal MRimaging unit (“microSigna 7.0” collaboration between GE Health-care[Waukesha, Wis.] and Varian [Walnut Creek, Calif.]) using asingle-channel transmit-receive partial birdcage radiofrequency coil.Sagittal MR images of the cell samples were obtained with a fastspin-echo sequence (3000/30) and a multiple-echo spin-echo sequence(4000/15, 30, 45, 60), using a field-of-view of 3.5 3 3.5 cm, a matrixof 256 3 256 pixels, and a section thickness of 0.5 mm. Pixelwise T2relaxation time maps generated by using custom research software(Cinetool; GE Global Research Center, Niskayuna, N.Y.) were used tomeasure T2 relaxation times of each sample through operator-definedregions of interest. Following MR imaging, the chondrogenic potential ofthe cell samples was evaluated.

In Vivo MR Tracking of Ferumoxytol-Labeled MSCs

Next, in vivo-labeled MSCs were implanted into osteochondral defects ofknee joints of seven recipient rats (14 knees). Osteochondral defectswere created in the distal femoral trochlear groove of both knee jointsby using a microdrill (Ideal, Sycamore, Ill.). In each rat, 1×10⁶ invivo ferumoxytol-labeled MSCs in an agarose scaffold were implanted intothe right femur and 1×10⁶ unlabeled MSCs in an agarose scaffold wereimplanted into the left femur. MSC transplants were evaluated with MRimaging immediately after stem cell transplantation (n=7), as well as 2weeks (n=7) and 4 weeks (n=6) after transplantation, by using the sameMR technique described above. T2 relaxation time maps were generated.After the last MR image was obtained, at 2 weeks (n=1) and 4 weeks (n=6)after transplantation, animals were sacrificed, and specimens wereprocessed for histopathologic correlations, which includedhematoxylin-eosin, 3,3′ diaminobenzidine-Prussian blue, and Alcian bluestaining Immunohistochemical staining against CD105 (Endoglin M-20;Santa Cruz Biotechnology) and CD68 (Abcam) were performed to evaluateMSCs and macrophage populations in osteochondral defects, respectively.

Statistical Analysis

T2 relaxation times and iron uptake data were compared for significantdifferences between different experimental groups by using t tests.Within each group, changes in MR data over time were examined by usingordinary least squares linear regression analyses. The t tests, analysisof variance, and linear models were computed by using the t test and theaov and lm functions in R (version 2.15.2) respectively. Because theright and left knees of each rat contained different implants, it wasassumed that MR images of each rat's knee were independent observations.To examine the possibility that data from the same rats were dependent(e.g., different rats metabolized the iron labels at different rates),multilevel models were fit to MR data by using the R package lme4(version 0.999999-0), with specifications identical to each linearmodel. A variable that identified each rat was added as a random effect,and the fit of each model was compared. In each case, the model fitswere not significantly different. For all analyses, a P value of lessthan 0.05 was considered to indicate a significant difference amongdifferent experimental groups or different times of observation.

Results

MSC Immunostaining

The yield from bone marrow aspirates was approximately 400 million cellsfor both ferumoxytol-injected animals and untreated control animals.MSC-selective culture led to separation of MSCs (attached to the flask)from other cells (in solution). At day 7, approximately 5 million cellsremained attached to the flask. Staining in a mean of 181.3 day 7 cellsper high-power field 6 7.9 (standard deviation) (70.2% 6 1.9) waspositive for CD105, while staining in only a mean of 33.4 day 7 cellsper high-power field 6 5.6 (13.5% 6 2.3) was positive for CD68 (FIG. 3).Of note, freshly extracted MSCs are small in size and slowly expand inculture. Expansion in cell culture is needed prior to ex vivo labelingto achieve satisfactory cell survival. Ex vivo labeling requires 4 hoursof fetal bovine serum deprivation, exposure to a transfection agent(protamine), and multiple centrifugation steps that freshly extractedcells can hardly withstand (FIGS. 3-4).

Evaluation of Ferumoxytol Uptake by MSCs

MSCs labeled with FITC-ferumoxytol demonstrated cellular iron oxideuptake at fluorescence and confocal microscopy, without apparentdifferences in cytoplasmic nanoparticle compartmentalization between invivo- or ex vivo-labeled cells (FIG. 4). Electron microscopy localizediron oxide nanoparticles in secondary lysosomes (FIG. 4). However,confocal and electron microscopy examinations revealed a higher quantityof iron oxide nanoparticles in in vivo-labeled MSCs compared with exvivo-labeled MSCs (FIG. 4, FIG. 9). The fluorescence intensity of invivo-labeled cells (Δ intensity=47.025) was 3.2 times higher comparedwith the fluorescence intensity of ex vivo-labeled cells (Δintensity=14.527, P=0.00005). The 3D plots, representingFITC-ferumoxytol concentrations as peaks per pixel on the area ofindividual cells (FIG. 4) confirm that labeled in vivo cells are smallerin size and contain more iron nanoparticles than labeled ex vivo cells.Both labeling techniques showed significantly higher fluorescenceintensities as compared with control cells (P<0.001) FIG. 4).

Evaluation of in Vitro MR Signal Intensity Effects ofFerumoxytol-Labeled MSCs

In vivo-labeled MSCs displayed strong signal intensity effects onT2-weighted MR images with significantly shortened T2 relaxation times(mean, 8.292 msec+/−6 2.326) compared with un-labeled control cells(mean, 33.614 msec 6 5.111; P=0.024) (FIG. 5A). Follow up studiesdemonstrated a slow decline in T2 signal intensity effects of labeledMSCs over time, which corresponded to a slow decline in cellular ironcontent (FIGS. 5A-C). After 3 weeks of cell culture, the T2 signalintensity of in vivo-labeled MSCs was not significantly different fromthat of unlabeled control cells (P=0.167) (FIG. 5B). A two-way analysisof variance confirmed that differences between groups (F=52.75; df=1,20; P=0.00000002) and between weeks (F=20.99; df=1, 20; P=0.00006) andthe interaction between the two (F=6.29; df=1, 20; P=0.017) were allsignificant. Accordingly, the iron uptake per cell, as measured byinductively coupled plasma optical emission spectrometry, wassignificantly higher for in vivo-labeled MSCs at day 7 (at day oftransplantation, mean was 4.276 pg per cell+/−0.190) compared withunlabeled cells (mean, 0.490 pg per cell+/−0.063; P, 0.0001) and exvivo-labeled cells (mean, 1.877 pg per cell+/−6 0.183; P<0.0001) (FIG.5B). Labeled cells at day 14 showed significantly higher iron uptakethan unlabeled cells (P=0.02) but not ex vivo-labeled cells (P>0.05)(FIG. 5B). In vivo-labeled MSCs and unlabeled control cells showed nodifferences in chondrogenic differentiation (FIG. 6).

In Vivo MR Tracking of Ferumoxytol-Labeled MSCs

MSCs from ferumoxytol-treated donors, transplanted into osteochondraldefects of recipient rats, showed strong signal intensity effects onT2-weighted MR images with significantly shortened T2 relaxation times(mean, 15.459 msec+/−0.729) compared with unlabeled control cells (mean,24.423 msec+/−1.213 P=0.0002) (FIG. 7). Longitudinal follow-up studiesrevealed slowly decreasing T2 signal intensity effects of unlabeledcontrol cells over time, apparently because of local cell proliferationand decreasing proton (water) content of the scaffold. Conversely, theT2 signal intensity effects of iron-labeled cells remained stable overtime, with the co-efficient on week, β_(week), of 20.465 msec+/−0.311(P=0.152), which may be due to combined effects of decreasing scaffoldproton (water) content and slow iron metabolism over time. T2 relaxationtimes of iron-labeled MSC transplants were significantly lower comparedwith those of unlabeled control cells at all times of observation(P<0.05) (FIG. 7), although the difference between labeled and unlabeledcells decreased slowly during 4 weeks. A two-way analysis of varianceconfirmed that differences between groups (F=17.14; df=1, 20; P=0.0005)and between weeks (F=5.60; df=1, 20; P=0.028) and the interactionbetween the two (F=6.64; df=1, 20; P=0.018) were all significant. Apower analysis indicated that future validation studies will need atleast five samples in each treatment group to achieve 80% power by usinga two-sample t test to detect an effect at week 2 and at least eightsamples per treatment group to detect an effect at week 4.

Corresponding 3,3′ diaminobenzidine-Prussian blue stains confirmeddecreasing iron staining of labeled MSC transplants over time,indicating slow iron metabolization (FIG. 8). Hematoxylineosin stainingin histopathologic examinations demonstrated engraftment offerumoxytol-labeled MSCs in the osteochondral defect, without anynotable morphologic difference, compared with unlabeled control cells(FIG. 8). At 4 weeks after MSC implantation, both labeled and unlabeledimplants had started to remodel the defect and to produce a chondrogenicmatrix, as evidenced by staining that was positive for Alcian blue (FIG.8). A stain that was positive for CD105 for both week 2 and week 4implants confirmed implantation of MSCs (FIG. 10). Immunohistochemicalstains revealed staining that was negative for CD68 for week 2 implantsbut slightly positive for CD68 surrounding the defect in week 4implants, suggesting minimal host macrophage influx (FIG. 10).

Synthesis of FITC-Conjugated Ferumoxytol

The carboxydextran coated ferumoxytol nanoparticles were firstcross-linked with epichlorohydrin for better stability in vivo asdescribed previously (93), then dialysis to remove low molecular weightcompounds against water using dialysis tubing (12-14K cutoff) over threedays yielded cross-linked iron oxide nanoparticles (CLIO). The obtainedamine-presenting nanoparticles in PBS buffer were then reacted with aDMSO solution of Fluorescein isothiocyanate (1:8 CLIO:FITC molar ratio).Purification with Microcon® centrifuge filters (¹⁰K cutoff, 5 mL→0.2 mLvolume reduction, 4600 rpm, PBS buffer addition and centrifugation) wasrepeated 10 times until the filtrate had no fluorescence to afford apurified product CLIO-FITC. Each nanoparticle on average had 3.8Fluorescein molecules. The amount of FITC covalently linked to ananoparticle was calculated using two methods. In the first method, FITCconcentration was determined by subtracting the maximum absorption (492nm) of CLIO-FITC from the absorbance of unconjugated TNP alone (measuredfor CLIO-NH2 at the same concentration of iron) and dividing the resultby known extinction coefficient of FITC (70,000 M⁻¹ cm⁻¹) at 492 nm. Inthe second method, the FITC's emission peak of a diluted (to avoidfluorescence self-quenching) CLIO-FITC was integrated and itsconcentration was estimated using a calibration plot obtained for a setof standard FITC solutions. Both methods gave consistent results (lessthan 8% difference) for three different solutions of CLIO-ICT.

Remarks

It is noted that the in vivo method does not use any additionaltransfection agents as is common in ex vivo approaches. In other words,MSCs efficiently phagocytose ferumoxytol without transfection agents invivo. However, it is noted that MSCs do not phagocytose ferumoxytol exvivo with high enough efficiency to enable in vivo tracking with MRimaging.

It is still further noted that the in vivo method does not require anyex vivo manipulations to the harvested stem cells. This is important fortranslational efforts.

It is further noted that other studies showed uptake of iron oxidenanoparticles by bone marrow macrophages in animal models and inpatients. These iron-labeled macrophages migrate into apoptotic stemcell transplants, which can be used for detection of stem cell deathand/or rejection. Macrophages migrate to much lesser extent into viabletransplants, which was below detection limits of our cellular MR imagingtest.

It is still further noted that the in vivo method does not use any exvivo labeling as is common in ex vivo methods of stem cell labeling.

It is still further noted that the in vivo method eliminates risks ofcontamination and biologic alteration of bone marrow-derived stem cellscaused by ex vivo-labeling procedures and that the method could beimmediately applied in a clinical setting for in vivo tracking of bonemarrow-derived stem cells in arthritic joints or other target tissues.

What is claimed is:
 1. A method for in vivo and noninvasive monitoringof stem cell implants, comprising: (a) intravenously injecting ironoxide nanoparticles into a subject to achieve in vivo phagocytotic ironlabeling of stem cells; (b) harvesting from bone marrow of said subjectthe in vivo iron labeled stem cells; (c) directly after harvesting,without ex vivo iron labeling of the stem cells, transplanting harvestedin vivo iron labeled stem cells into a different subject; and (d)monitoring in vivo and noninvasively the transplanted in vivo ironlabeled stem cells using magnetic resonance imaging.
 2. The method asset forth in claim 1, wherein the stem cells are mesenchymal stem cells.3. The method as set forth in claim 1, wherein the harvesting takesplace in about one to three days from the intravenous injection.
 4. Themethod as set forth in claim 1, wherein the intravenous injection of theiron oxide nanoparticles is dosed at 28 mg of iron per kg of body weightof the subject.
 5. The method as set forth in claim 1, wherein theintravenous injection of the iron oxide nanoparticles is dosed at 5-10mg of iron per kg of body weight of the subject.
 6. The method as setforth in claim 1, wherein the harvested stem cells are transplanted inan organ of a different subject.
 7. The method as set forth in claim 1,wherein the harvested stem cells are transplanted in a joint, a brain, aheart, a liver or a pancreas of a different subject.
 8. The method asset forth in claim 1, wherein the in vivo phagocytotic iron labeling ofstem cells occurs in vivo without the use of a transfection agent. 9.The method as set forth in claim 1, wherein the method does not use anyex vivo labeling or ex vivo manipulations to the stem cells.